World ethanol production totalled 46 billion litres in 2005 and is rapidly increasing (EU commission, 2006). The production of ethanol can be either from starch or sugar, which primarily consist of glucose or from lignocellulosic material such as wood, straw, grass, or agricultural and household waste products. The main constituents of lignocellulosic material are the polymers cellulose and hemicellulose. While cellulose is a rather homogenous polymer of glucose, the hemicellulose is a much more complex structure of different pentoses and hexoses. The complex composition of hemicellulose requires different means of pre-treatment of the biomass to release the sugars and also different fermenting organisms. To produce ethanol by fermentation a microorganism able to convert sugars into ethanol rapidly and with very high ethanol yields is required. Traditionally, organisms such as the yeast Saccharomyces cerevisiae or the bacterium Zymomonas mobilis have been used, but these organisms have limitations especially when it comes to fermentation of the pentose sugars from hemicellulose and the risk of contamination.
Lignocellulosic material is the most abundant source of carbohydrate on earth, and the second most important sugar in this biomass is xylose—a pentose sugar. If production of ethanol from lignocellulosic biomass is to be economically favourable, then all sugars must be used, including pentoses.
Thermophilic anaerobic bacteria have proven to be promising candidates for production of ethanol from lignocellulosic materials (WO 2007/134607). The primary advantages are their broad substrate specificities and high natural production of ethanol. Moreover, ethanol fermentation at high temperatures (55-70° C.) has many advantages over mesophilic fermentation. One important advantage is the minimization of the problem of contamination in continuous cultures, since only few microorganisms are able to grow at such high temperatures.
WO 2007/053600A describes how close to stoichiometric yields of ethanol from glucose and xylose can be obtained by deleting the genes coding for lactate dehydrogenase, phosphotransacetylase and acetate kinase in Thermoanaerobacterium saccharolyticum. However, this approach may not be applicable in thermophilic organisms having multiple phosphotransacetylase and acetate kinase genes and does not facilitate utilization of glycerol.
Ethanol yield is of great importance for the production economy of bioethanol, since increased income can be obtained without an increase in biomass price or production costs. For Escherichia coli it has been shown that once the enzyme levels and substrate are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting for alcohol yield (Berrios-Rivera et al., 2002).
It has been shown that addition of glycerol to the growth medium of certain Clostridia can increase the production of alcohols (Vasconcelos et al., 1994). However, optimal alcohol production was achieved at a glycerol/glucose ratio of 2, and glycerol is therefore considered to be a major expense.
A glycerol dehydrogenase gene has been introduced into Escherichia coli to promote the production of 1,2-propanediol (Berrios-Rivera et al., 2003) and into Clostridium acetobutylicum to promote production of 1,3-propanediol (Gonzalez-Pajuelo et al., 2006). In both cases the glycerol dehydrogenase is in the direct pathway to the produced propanediol, and no production of propanediol occurs without the presence of the gene. The major function of the glycerol dehydrogenase is not to change the redox balance of the cell, but rather to provide a new pathway.
It is therefore one object of the present invention to provide recombinant bacteria, in particular thermophilic anaerobic bacteria, with increased ethanol production capabilities which are capable of overcoming the above mentioned obstacles.